Inhibition of platelet aggregation by fibronectin

BIOCHEMICAL

Vol. 116, No. 1,1983 October

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 135-140

14, 1983

INHIBITION OF PLATELET AGGREGATION BY FIBRONECTIN Samuel

A. Santoro

Division of Laboratory Medicine Washington University School of Medicine St. Louis, Missouri 63110 Received

August

15,

1983

SUMMARY: Ffbronectin is a large dimeric glycoprotefn found in plasma, on cell surfaces and as a component of the extracellular matrix which has been implicated in a variety of adhesive processes. The role of ffbronectin in platelet function has not been clarified. The present investigation demonstrates that an excess of exogenously added ffbronectin inhibits platelet aggregation induced by either thrombfn or A83187 at a step subsequent to platelet activation and secretion. Similar concentrations of fibrinogen or von Willebrand factor, both of which also bind to the surface of activated platelets, are not inhibitory. These results are consistent with the concept that ffbronectin is one of the important mediators of platelet aggregation.

Fibronectin surfaces

is a large

and as a component

reviews

of this

important

has been implicated and spreading,

exists

as higher

appear

to be mediated cell

functional While processes fibronectin

molecule

motility,

fibronectin

exists

surfaces, domains

by the ability proteoglycans

with

subsequent

lysis

in plasma,

on cell

matrix.

Several

excellent

appeared

(l-4).

Ffbronectin

including

adhesiveness

phenotype dfmer;

The diverse

the cell

functions

of fibronectfn and other

and opsonizatfon.

via

form

of ffbronectfn

to interact

proteins

surface

specifically

several

discrete

(41.

above

supporting

collagen

a role

for

as accumulated,

fibronectin

well

of platelets

and colleagues (6) suggested

collagen-induced by sonicatfon that

fibronectin

135

supporting

developed.

as one of the several after

in the biologic

the evidence

has been less

of ffbronectin

associated

of morphologic

(51.

found

functions

as a dfsulfide-bonded

in hemostasis

identification

of cellular

polymers

the evidence outlined

have recently

control

order

glycoprotein

of the extracellular

in a variety

Plasma

with

adhesive

Following

proteins

platelet and detergent

a role

for

the

remaining

aggregation treatment,

was the collagen

receptor

and Bensusan on

0006-291X/83 $1.50 Copyri&ht 0 I983 by Academic Press, Inc. All rights of reproduction in any form reserved,

Vol. 116, No. 1, 1983

platelets.

BIOCHEMICAL

More definitive

Recent

demonstrations

activation surface

of unactivated

surface

of activated

accompanied suggest

(7,8)

fibronectin

is

or collagen

(9),

platelets platelets

(10,ll)

fibronectin

supported

secreted

from platelets

that

fibronectin

is

but

becomes

and that

platelet

of specific

may play

RESEARCH COAWdJNlCATlONS

have not

(7,10,11)

by the development

that

platelet

studies

that

by thrombin

AND BIOPHYSICAL

following

not

present

expressed

in

on the

on the

binding

role

proposal.

activation

fibronectin

a significant

this

is

sites

the later

(12)

steps

all of

aggregation. MATERIALS AND METHODS

- Gelatin and the ionophore A23187 were obtained from Sigma. Materials C14]serotonin (50nCi/mmol) was purchased from Amersham. Purified human thrombin with a specific activity of 3390 U/mg was generously provided by Sepharose 48 and cyanogen bromide-activated Sepharose Dr. Joseph P. Miletich. 48 were obtained from Pharmacia. Fibrinogen (grade L) was obtained from Kabi. Von Willebrand factor was purified from cryoprecipitate as described (13). Purification of fibronectin - Fibronectin was purified from human plasma by attinity chromatography on gelatin-Sepharose 4B (14). Plasma was made 2mM in PMSF, diluted with an equal volume of 50nM Tris (pH 7.6), 2.5nM EDTA, 0.15M NaCl and applied to a Sepharose 43 column equilibrated with the above buffer to remove material which binds to the Sepharose matrix prior to chromatography on gelatin-Sepharose 48. Fibronectin was eluted from the gelatin-Sepharose 48 with 4M urea (14). Platelet aggregation and secretion - Washed platelets were prepared by centrifugation as described earlier (13). For use in the present studies, the platelets were resuspended in 0.14M N&l, 2.7mM KCl, 12mM NaHC03, 0.42mM NaH2P04, 0.55mM glucose, 5n1M HEPES (pH 7.35), 0.3% bovine serum albumin at a platelet count of 6 x IO8/ml. Aggregation studies were performed using a Payton dual channel aggregometer. The reaction mixture typically consisted of 150~1 of the platelet suspension and 25OPl of 0.194 NaCl, 0.05M sodium phosphate (pH 7.4) containing fibronectin or fibronectin domains. After mixing and incubation at 37 degrees for 2 min with stirring at 950 rpm in the reaction cuvette, the sample was made 2mM in CaC12 and the aggregating agent added. Thrombin was added in a volume of 30cll to give a final concentration of 0.1 U/m?. A23187 dissolved in Me2SO was added in a volume of 10~1 to give a final concentration of 2wM A23187. Serotonin secretion studies were performed as recently described (16) using washed platelets which had been preloaded with CI4Clserotonin. RESULTS AND DISCUSSION This

investigation

fibronectin

and,

involved

in platelet

between large secreted

relative

or plasma

based

on the

by implication,

platelets excess

is

also

aggregation, by virtue

of its

to platelet proteins

involved

underlying

assumption

platelet-secreted

the molecule divalent surface

136

if

fibronectin,

functions structure.

components

in platelet

that

to form

are bridges

When present and other

aggregation

plasma

in

a

plateletone would

predict

Vol.

116,

that

No.

1983

1,

fibronectin

which

BIOCHEMICAL

would

become

immune complexes

This

condition

potent are

thrcmbin

each capable

proteins

in a simple,

As shown in extent

of platelet

increased.

thrombin

is

inhibition

Complete

aggregometer correspond

aggregation

tracings, to the unlikely

binding,

0

loss that the

these

conditions

occurs

of the

by two

these

agents

of exogenously the desired

a progressive

at approximately

of aggregation, achieved. second

the observed proteolytic

Since

as the concentration

is achieved

not

excess.

induced

A23187.

to achieve

in

added

excess

of

system.

inhibition is

of antibody

in the absence

is possible

to that

aggregation

ionophore

well-defined

analagous

conditions

platelet

platelets it

1, under

Maximal

fibronectin.

It

Fig.

under

and the calcium

of aggregating

RESEARCH COMMUNICATIONS

in a manner

by studying

such as fibrinogen,

fibronectin

inhibitory

may be soluble

was achieved

agents,

AND BIOPHYSICAL

action

109 167)

are

!iOOpg/ml

attributable

of thrombin

or other

the is

of

by the

of inhibition

phase of thrombin-induced

in

of fibronectin

as reflected

The extent

results

inhibition

appears

to

aggregation. to inhibition early

of

event

in

b

581 (51)

I

Ffg. 1. Inhibition of platelet aggregation by fibronectin. The reaction mixture consisted of 150~1 of the washed platelet suspension (6 x 108 platelets/ml), and 250~1 of 0.05M sodium phosphate (pH 7.41, 0.15M NaCl containing various concentrations of fibronectin. The suspension was made in CaC12 and either thrombin (O.lU/mll or A23187 (2pM) added to initiate aggregation. The uppermost number over each aggregation profile indicates final concentration lug/ml of fibronectin. The number in parentheses 1 indicates the percent of [ 4Clserotonin secreted from the platelets in response to the stimulus. Aggregation induced by thrombin (a), Aggregation induced by A23187 (b). 137

ZmM the

Vol. 116, No. 1, 1983

the action

of thrombin

effectively (Fig.

since

and A23187

fibronectin

which

of aggregation

activation

and secretion.

consistent

small

with

After

apparent,

characteristic roles

These of a large

inhibit

because (121,

but surface It

is

maximal

to the presence

of a high

glycoprotein.

aggregation

As shown in

interesting

inhibition the

saturation

of the

(18,191

Fig.

to those

effect.

at the platelet

of small

concentration

2, concentrations

of fibronectin

Furthermore, is unlikely

surface

since

and von Willebrand

which

the observed to arise

not only factor

simply

fibronectin (201

bind

platelets. to note

of platelet

that

specific

the concentration

aggregation

same concentration

is consistent

similar

by fibronectin

fibrinogen

of activated

the

change

may play

aggregates

were without

both

fibronectin

larger

into

no

the shape

the association

aggregation

also

that

were

and in

factor

essentially

observation

not due simply

hindrance

appears

the

aggregates

had undergone

aggregates

or von Willebrand

of steric

using

is

aggregation.

added

of platelet

small

experiment This

observed

even these

Thus it

formed

platelet

are

platelet

inhibition

of aggregation

platelet

initially

exogenously

of fibrinogen

of 4-8 platelets.

(17).

findings

aggregation

composed

clearly

the

of platelet

aggregates

standing

by

to platelet

the aggregation

of activation

of full

subsequent

inhibition

also

of

that

following

the platelets

are

is clear

insnediately

inhibition

small

11, it

the

induced

by concentrations

at a step

although

which

characteristic

occur

prolonged

in stabilizing

aggregates

the

platelet

inhibited

is

of fibronectin

of C14C]serotonin

(Fig.

confirmed

the partial

aggregometer. longer

must

aggregation

concentrations

secretion

aggregation

Examination

only

identical

the

RESEARCH COMMUNICATIONS

A23187-induced

not markedly

examinatfon

by fibronectin. revealed

is

inhibit

inhibition

Microscopic

since

by essentially

Furthermore,

thrombin

AND BIOPHYSICAL

on platelets

inhibited

1).

both

BIOCHEMICAL

at which

fibronectin with

in

binding

the hypothesis 138

of fibronectin

the present

investigation

Plow and Ginsberg sites that

giving

(12)

is observed

on platelets. the

inhibition

This of

to

BIOCHEMICAL

Vol. 116, No. 1. 1983

AND BIOPHYSICALRESEARCH

COMMUNICATIONS

1 +a

r

:

4

Fw

Specificity of inhibition of platelet aggregation by fibronectin. Exper mental conditfons were as described in the legend to Fig. 1. Effects on thrombin-induced aggregation are shown in panel a. tracing 1 - aggregation in the absence of exogenously added proteins; tracing 2 - lack of effect of 447ug/ml of von Willebrand factor; tracing 3 - inhibition by 543pg/ml of fibronectin. The effects on A23187 - induced aggregation are shown in panel b. Tracing 1 - aggregation in the absence of exogenous protein; tracing 2 - aggregation in the presence of 581ug/ml fibrinogen; tracing 3 aggregation in the presence of 47gPg/ml von Willebrand factor; tracing 4 inhibition of aggregation by 581pg/ml fibronectin.

aggregation

by high

of each of the thus

fibronectin

precluding

the inhibition Although

noted for

that

fibrinogen

the affinity

fibrinogen) for

an important patient

is

whose

with

in platelet was unable

Cohen et al

which

of magnitude

in

the

present

candidate,

fibrin less

The present

than

aggregation

is

to support by the addition

is not et al

platelets is

that

platelet

fibronectin

(21)

and

greater

than

that

of

fibronectin

by the description

of purified

exogenous

study

the affinity

supported normal

to produce

Ginsberg

(which

contention

139

molecule

interacts

to afibrinogenemic for

that

the occupation

fibronectin

fibronectin

seem a likely

(23)

from

bridges.

observed

of fibronectin

was corrected with

by a dimeric

of fibronectin

two orders

plasma

The abnormality in agreement

would

the platelet.

role

results

of interplatelet

aggregation

the binding

fibronectin

sites

of the components of platelet

have observed

of fibronectin

binding

the formation

The identity

clear.

concentrations

aggregation fibronectin. is

not

plays of a (22). We are required

Vol. 116, No. 1, 1983

for (91,

platelet this

aggregation. is

implicated are

all

BIOCHEMICAL

not

Since

unexpected.

in platelet at least

the multiple

formation

gives

rise

secrete

It is probably

of these

proteins

to interactions

platelet

RESEARCHCOMMUNICATIONS fibronectin

significant

fibrinogen,

It seems likely

divalent.

of stable

platelets

aggregation,

interactions

one another

AND BIOPHYSICAL

that

thrombospondin that

a complex

with

the platelet

of sufficient

upon activation

strength

the proteins

now

and fibronectin matrix

formed

surface

by

and with

to result

in the

aggregates. ACKNOWLEDGMENTS

I thank Joseph P. Miletich Cowan for technical assistance This investigation typescript. grant-in-aid from the American part by the Missouri affiliate.

for the gift of purified thrombin, Joseph F. and Paula J. Game1 for preparing the was supported by NIH grant HL 29608 and by a Heart Association with funds contributed in REFERENCES

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